Methods and apparatus for measuring the light absorbance of a substance in a solution

Abstract

Disclosed is an apparatus for measuring the absorbance of a substance in a solution, comprising: i) a sample cell (30) of known path length (b) for containing said solution (S), said cell being transparent to light of a predefined wavelength spectrum; ii) plural LED's each being independently operable by means of a controller (25) each for emitting light, within said predefined wavelength spectrum, along a light path; iii) a band pass filter (22) in the light path; iv) a beam splitter (24) for dividing light from said source propagating along the path into a first portion and a second portion, said first portion being directable by the beam splitter toward a reference detector (42) and said second portion being directable into the cell (30); v) a reference detector (42) for detecting the intensity of said first portion of light directed by said beam splitter; and vi) a sample detector (40) for detecting the intensity of the second portion propagating from the cell; the apparatus allowing a sample in the cell to be inexpensively subjected to more than one wavelength of light for quicker or more accurate analysis.

Description

[0001]The present invention relates to methods and apparatus for measuring the absorbance of light of a substance in a solution using visible or non visible light, the substance having a capacity to absorb light at a specific wavelength or wavelengths. The methods and apparatus have particular utility in detecting the concentration of proteins and nucleic acids for example where such proteins or nucleic acids are refined during liquid chromatography.FIELD OF THE INVENTION[0002]The invention relates to methods and apparatus for measuring the absorbance of a substance in a solution, typically a substance exhibiting UV light absorption at a wavelength of 400 nm or less.BACKGROUND OF THE INVENTION[0003]Many substances absorb ultra violet or visible light due to their chemical composition. The absorption of light by substances has been used as the basis for detecting the presence of, and measuring the concentration of, such substances for many years. The concentration of the substance ca...

AI Technical Summary

Benefits of technology

The present invention relates to a method and apparatus for measuring the light absorbance of a substance in a solution, which can be used to determine the concentration of the substance without needing to know its exact molar absorptivity. This involves transmitting light of a certain wavelength through the substance and quantifying the intensity of the light that comes out, using a second LED light source with a different wavelength output. The method and apparatus can be used for a variety of substances such as proteins, peptides, carbohydrates, lipids, nucleic acids, and xenobiotics. This invention offers a non-destructive and efficient way to measure the concentration of substances in solutions.

Problems solved by technology

Mercury lamps have relatively short lifetimes and can prove unstable with time; furthermore, the disposal of these lamps can lead to environmental problems.

The other lamps used to generate ultra violet light, such as the deuterium and the xenon flash lamps, disadvantageously require high voltages, need complicated electronics and often prove unstable with time.

All of the currently used ultra violet light sources are relatively large and are consequently unsuitable for miniaturisation of analytical instruments.

Moreover, all of the lamps generate significant amounts of heat due to the high voltages required for their operation.

However, although detection systems employing LEDs are disclosed, there are no experimental data to indicate that the proposed systems were indeed successfully employed to measure nucleic acid levels in polymerase chain reaction assay.

Furthermore, the system lacks a band pass filter which acts to both narrow the bandwidth and block light in the visible region of the spectrum.

However, no experimental data that show the linearity and dynamic range are provided.

However, while this may be sufficient to measure ozone concentrations, the lack of a band pass filter which can reduce the band width to, for example, one tenth of the half value width of the ozone absorption peak will significantly reduce the linearity and dynamic range of the detector (Practical Absorbance Spectrometry. Ed. A Knowles and C. Burgess, Chapman and Hall, New York).

This system also lacks a reference photo detector, so no measurement of the intensity of the emitted light is made.

This means that compensation of variations of the emitted intensity due to changes in temperature is not possible.

However, known LEDs have only a limited light wavelength output range.

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